Rotaviruses are the major cause of life-threatening diarrheal disease in infants and animals worldwide. The long-term research objective of this laboratory is to understand, in detail, the molecular biology of rotavirus protein function as it relates to mechanisms of pathogenesis and virus assembly. These complex viruses, that lack an envelope, have a unique morphogenetic pathway involving immature particle budding through the membranes of the endoplasmic reticulum (ER). Studies of the role of the nonstructural glycoprotein, NSP4, in viral morphogenesis led to our discovery that this protein affects calcium homeostasis and functions as an enterotoxin. NSP4 plays a key role in rotavirus pathogenesis by triggering a cell signaling pathway that results in diarrhea. A cleavage product of NSP4 secreted into the medium of virus-infected cells binds to uninfected cells and triggers a IP3-mediated, PLC-dependent signaling pathway leading to elevated [Ca2+]i and chloride secretion as well as alterations in polarized epithelial cell integrity. Intracellular NSP4 triggers a distinct PCL-independent signaling pathway and regulates virus replication, through mechanisms that remain to be characterized. Antibody to NSP4 induces protective immunity from rotavirus-induced diarrhea in mice. Other recent data indicate multiple pools of NSP4 exist and knockdown of NSP4 expression leads to alterations in the accumulation and distribution of almost all other rotavirus proteins and modulates viral transcription. Thus, NSP4 is an important virulence factor, and viral-induced cell signaling plays previously unrecognized role(s) in rotavirus replication, morphogenesis and pathogenesis. This grant application requests support for continuation of an ongoing program to gain mechanistic insight into functional domains responsible for the pleiotropic properties of NSP4. We seek to dissect the molecular details of enterotoxin- and RV-cell interactions related to viral replication, morphogenesis, and pathogenesis. The specific aims of the proposed work are: (1) To understand the forms of expressed intracellular NSP4 (iNSP4)and their effects on epithelial cell function; (2) To determine if iNSP4-induced elevations of [Ca2+]i are essential for the effects of iNSP4 on viral RNA synthesis and protein distribution; and (3) To dissect the pleiotropic properties of NSP4 by understanding the binding of extracellular NSP4 (eNSP4) to the l-domain and by studying the role of particle-associated NSP4 (pNSP4) in virus entry and infection. These studies will provide a more detailed molecular understanding of rotavirus replication, pathogenesis and viral budding through ER membranes. Understanding these unique aspects of rotavirus pathogenesis offers opportunities to develop new strategies to prevent and control rotavirus disease in children and animals, and to understand the fundamental autophagy and exocytic processes of polarized intestinal cells. [unreadable] [unreadable] [unreadable]